Project Abstract This submission is in response to NOT-AI-19-040, ?Administrative Supplements for Characterization and Further Development of Humanized Immune System Mice. This special interest opportunity is of particular importance to the outcome of our parent R24 grant, due to the inconsistency of engraftment of mobilized peripheral blood (mPB) units, and the lack of development of all components of the human immune system in the mice. The goal of Aim 3 in our R24 grant is to humanize our new strain of immune deficient HD mice in order to evaluate the role that the human immune system has on the course of the disease. If mPB units fail we waste funding and important mice. If the mice have an incomplete human immune system the impact of our R24 grant outcomes will be less striking. In this supplement we will test two hypotheses, using NRG and NSG.A2 mice; 1) Optimal donors of mobilized peripheral blood (mPB) units can be proactively identified by a blood test prior to mobilization, giving rise to consistent batches of humanized mice for HIV and other studies, and 2) Co-transplantation of the human stem cell graft with human induced pluripotent stem cell-derived endothelial cells engineered to secrete IL-7 and IL-15 will enhance the TCR repertoire and broaden human immune system development. By validating the HLA type, CD47 levels (responsible for mouse phagocytosis of human cells), selectins, fucosylation levels and other characteristics of the donor mPBSC units that give the most consistent and robust engraftment levels, we can help the field by establishing optimal donor criteria. For the planned co-transplantation studies, in comparison to the iPSC-derived IL-7/IL-15 producing endothelial cells, we will use endothelial progenitors expanded from the same mPB unit used for CD34+ cell HSC isolation. The endothelial cells will engraft into the newborn mouse pup thymus, mucosa, and other lymphoid organs to allow better TCR selection from T cell progenitors developing from the transplanted HSCs. The sustained presence of IL-7and IL-15 will enhance overall human lymphoid cell development. Our goal is to significantly improve the consistency of engraftment of newborn immune deficient mouse pups with human CD34+ hematopoietic stem cells from mobilized peripheral blood units, increasing the range of human TCR development, and broadening immune responses by the human cells that have developed in the mice. This goal is highly relevant to our own work and to that of many others in the field.